Morphology of the oldest fossil subfamily of Limoniidae (Diptera, Architipulinae) in the light of exceptionally preserved Mesozoic material

Based on known fossil evidence the extinct subfamily Architipulinae is considered to be the oldest evolutionary group of the Limoniidae, the largest family within the infraorder Tipulomorpha. The morphology of this subfamily, which includes 11 genera, has so far been based mainly on wing venation. New well-preserved representatives of the genus Cretolimonia Kalugina, 1986 were recovered from the Jurassic/Cretaceous boundary of Shevia and Daya, Transbaikalia, as well as from mid-Cretaceous amber from Kachin, Myanmar. This new material enriches our knowledge of the subfamily Architipulinae and of the genus Cretolimonia, and allows us to ascertain the detailed morphological structure of the female copulatory apparatus with spermathecae and the structure of the male hypopygium. The combination of detailed impression fossils with a specimen preserved three-dimensionally in resin has permitted study of the morphology of this Mesozoic fly genus almost to the level of modern genera. The paper includes descriptions of four new species of Cretolimonia: C. lukashevichae sp. nov., C. pseudojurassica sp. nov., C. dayana sp. nov. from sedimentary rocks, and C. mikolajczyki sp. nov. from Myanmar amber, supported with a key to all known species.

The Architipulinae material described here shows an exceptional level of preservation, allowing us to augment the diagnosis of the genus Cretolimonia and enriching our knowledge of the whole subfamily Architipulinae. The hypopygium (a modified abdominal segment associated with the genitalia and having a clasping function in the males) is described for the first time on the basis of a perfectly preserved imprint in the rock, as well as the first representative of the subfamily from a fossil resin. Four new species of Cretolimonia are described, and a key to all known species is provided.

Amended diagnosis
The genus is distinguished from all Limoniidae by the characteristic wing venation pattern: vein Sc ends near Rs bifurcation, four radial veins present (R 1 , R 3 , R 4 and R 5 ), cross-vein r-r (R 2 ) atrophied, and vein R 3 very short, slightly curved; basal medial vein (Mb) well visible, long, all four medial veins present, d-cell closed, cross-vein m-cu located at the distal part of the d-cell base or sometimes in the middle (C. excelsa);   www.nature.com/scientificreports/ hooked at the end; inner gonostylus delicate, lobed; penis long, narrow, slightly sigmoidally curved; parameres broad at base, shorter than aedeagus (3D,4C). Female. Ovipositor short, slightly curved dorsally; three small spermathecae ( Fig. 3B, 4D).

Remarks
The male hypopygium is very well preserved in specimen No. 3063/1079 (Fig. 3D), being only slightly deformed by elongation of the left gonocoxite during fossilization. In previously described species of this genus the copulatory apparatus has not been preserved. www.nature.com/scientificreports/  www.nature.com/scientificreports/ Remarks The holotype specimen shows two well-preserved wings and a fragment of the thorax. The left wing is longer than the right one, most probably deformed (elongated) during the fossilization process. Such a phenomenon has been observed in different groups of insects 25,26 . The right wing retained its normal structure.
Material examined Holotype No. 3795/628 (Fig. 5B) Description Well-preserved specimen. Wing length 2.6 mm; width 1.2 mm; body length 2.8 mm. Head. Considerably wider than its length; antennae 16 segmented, scapus large, tubular half as long as its width, pedicel round, almost half as wide as scapus; flagellum 14 segmented, the first basal segment expanded in the lower part, the last small and rounded; bristles are present on all the flagellomeres, on the three basal segments bristles 2.5-3 times as long as the width of the segment, on the other flagellomeres bristles are shorter, 1.5-2 times as long as the width of the segment, on the last segment 3 or 4 short bristles (Fig. 7B, 8A). Palpi invisible. Thorax. Wing about 2.3 times as long as its width (Fig. 7C, 8B), additionally expanded in the anal field, vein Sc ends opposite bifurcation of Rs; cross-vein sc-r is about its length opposite the end of Sc; R 1 ends opposite bifurcation of R 2+3+4 into R 3 and R 4 , R 2 (r-r) fully atrophied; Rs about 1/4 longer than R 2+3+4 ; R3 short, slanting, less than 1/3 length of R 2+3+4 ; R4 only slightly shorter than R 2+3+4 ; four medial veins present, and vein M 1 2.5 times longer than petiola and twice as long as upper edge of d-cell; d-cell trapezoidal, small, constituting only 1/10 of wing length; crossvein m-cu at end of d-cell, just before M 3+4 bifurcates into M 3 and M 4 ; A 2 vein strongly waved. Legs long, delicate, tibial spurs absent. Abdomen. Hypopygium (Fig. 7D, 8C): gonocoxites long, narrow, with numerous bristles, at end bearing a bunch of long bristles; outer gonostylus strongly chitinized, long, narrow, strongly hooked at end; inner gonostylus delicate, lobed, elongate at the end; penis long, narrow, slightly curved; parameres shorter than penis, broad at base, similar to parameres in Cretolimonia dayana sp. nov..
Remarks Cretolimonia mikolajczyki sp. nov. is the first representative of the genus, and of the Architipulinae, found in fossil resin. Its excellent preservation permits detailed examination of morphological characters, especially the structure of the antennae and copulatory apparatus.  16 belong to the fossil family Archilimoniidae. Archilimoniidae was included as a subfamily within the family Limoniidae, which we consider erroneous since the wing venation more closely resembles that of Pediciidae, especially in  www.nature.com/scientificreports/ the radial field 16 . This being the case, the oldest known representative of the Limoniidae would be Architipula youngi Krzemiński, 1992 3 from the Late Triassic of North America (ca. 220 Ma) 6 used for age calibration of Tipulomorpha 8 . It should be noted that the first 100 million years of dipteran, and hence tipulomorph, evolution is documented only by impression fossils, mainly wings 28 .
The earliest examples of Limoniidae revealing three-dimensional structure have been recovered from Lower Cretaceous Lebanese amber, but such inclusions are very few in number [29][30][31] . The specimens described here are of paramount importance for understanding the morphology of Architipulinae (Fig. 7A), and thus shed new light on the early evolution of flies of the suborder Tipulomorpha. The male genitalia of these flies are usually so severely deformed during fossilization that their precise structure and spatial arrangement cannot be determined. Although the preservation of Cretolimonia dayana sp. nov. is very good, being an impression fossil, it is only possible to obtain information in two dimensions. Therefore, finding a male of the same genus (C. mikolajczyki sp. nov.) in mid-Cretaceous amber presented a unique opportunity to fully reconstruct the genital anatomy, and thus to verify the structure of the hypopygium of C. dayana sp. nov. (Fig. 3B,4D). In sedimentary rocks, the genitalia of females are much better preserved, as they are usually strongly chitinized; in favourable circumstances even spermathecae are visible, and sometimes also genital plates 26 . However, so far, the number of spermathecae has not been known for the oldest known Limoniidae, the subfamilies Architipulinae or Eotipulinae. The well-preserved material from the Daya site allowed us to reconstruct the genitalia of the female C. dayana sp. nov. and to determine the number of spermathecae. The spermathecae are identical to those of most species in the family Limoniidae.
Most genera of Limoniidae have a full set of five radial veins, including the R 2 vein, which takes the form of a cross-vein r-r. However, as far back as the Early Jurassic, a number of genera had appeared in which the cross-vein r-r (R 2 ) had disappeared (i.e., atrophied) and one of these is the genus Cretolimonia. The disappearance of this cross-vein is observed also in some other Limoniidae, for example, in the subfamily Chioneinae, in the modern genera Gonomyia Meigen, 1818 and Rhabdomastix Skuse, 1890. There are, however, significant differences in other sectors of the wing in these genera; only three medial veins are always present, and the cross-vein m-cu is located in the anterior half of the d-cell, usually near the bifurcation of M 3+4 into M 3 and M 4 .
New material described here enabled us to characterize the morphology of the oldest (at least in the geological sense), extinct group of the Limoniidae; these features will be of great importance when introduced to the phylogenetical analysis of this large dipteran family.

Materials and methods
Geological context. This study was based on impression fossils and an amber inclusion (Fig. 9). The rock material comes from two sites, Shevia and Daya, in the Shelopuginsky District of the Chita region of Transbaikalia (Russia). Shevia (Dain Formation), dated Early Cretaceous, is located on the right bank of the Shevia River, 3 km below Shevia village, 2 km above the confluence of the Shevia and Shiviinsky Bumulei Rivers. The Daya site is located on the left bank of the Daya River above the Shevia Valley. It exposes sediments of the Glushkovo Formation, which are imprecisely dated but likely to lie close to the Jurassic/Cretaceous boundary [32][33][34] .
The investigated amber inclusion derives from a former amber mine located near Danai (Tanai) Town (approximately at 26° 150″ N, 96° 340″ E) in the Hukawng Valley, state of Kachin in northern Myanmar. Radiometric U-Pb zircon dating of the volcaniclastic matrix of the amber produced an age of 98.79 ± 0.62 million years (earliest Cenomanian) 29,35 . Specimen repository. All the specimens studied in the course of this work are deposited permanently in publicly owned collections in national museums. The Myanmar amber inclusion, the holotype of C. mikolajc-

Methods
Specimens were studied using a Nikon SMZ25 stereomicroscope under reflected light, and photographs taken with a Nikon DSRi2 digital camera. The surfaces of rock specimens were wetted with 98% ethyl alcohol to improve the image contrast. Drawings were executed on the basis of the photographs with permanent referral to the specimens. The terminology of wing venation follows 11,27 . Geological ranges of subfamilies of the family Limoniidae are based on published data 1,3,29-31,37 .